Congestive heart failure is one of the most severe health problems in the USA. Regardless of the etiology of the disease, the development of congestive heart failure is associated with a progressive reduction in the contractility of the heart. These defects in human cardiocyte contractility contribute to disease progression but their underlying cellular and molecular basis is not well understood. This limited understanding of dysfunctional properties of diseased human myocytes has resulted from difficulty in obtaining high quality myocyte preparations. Recently, a technique has been developed for the isolation of "healthy" myocytes from explanted human hearts. The objective of the proposed experiments is to use these myocytes to define the basis of the cellular abnormalities that are present in the failing myocyte and determine which of these can be reversed when the failing heart is supported with a left ventricular assist device (LVAD). The working hypotheses of the application are that 1) contractility is abnormal in failing human ventricular myocytes because of defective excitation contraction coupling and reduced sarcoplasmic reticulum Ca loading. 2) these defects are reversible by LVAD support, and 3) the heart failure phenotype can be induced by either alpha1 adrenergic agonist or interleukin-1B. The specific aims of the proposal are 1) to determine the differences in electrical and contractile characteristics of human ventricular myocytes isolated from nonfailing, failing and LVAD-supported hearts. 2) Determine the respective roles of L-type Ca current, the coupling of L-type Ca current to SR Ca release and SR Ca loading in the defective contractility of the failing myocyte. 3) Determine if exposure of nonfailing human ventricular myocytes to either alpha1-adrenergic agonist or ILlb induces a dysfunctional physiological phenotype. These studies will employ myocytes isolated from human hearts using a novel techniques, which minimizes myocyte damage. A variety of cellular techniques including whole cell voltage clamp, single cell contractions, Ca imaging, and confocal microscopy to measure local SR Ca release will be employed. These approaches should allow the cellular basis of dysfunctional contractility in human heart failure to be reliably determined for the first time. This project will involve close collaboration with the other investigators at Temple and UCSF. This work will explore both the fundamental in-vivo derangements and their molecular basis. Defining the basis of dysfunctional contractility of human myocytes, processes that are reversible and signaling